Functional characterization of 3 novel NUDT15 risk variants. (A) Shown are the positions of novel (red text) and known risk variants (black text) in the coding region of the NUDT15 gene. (B) Shown is the spatial distribution of amino acid residues affected by risk variants in the human NUDT15 protein. Presented as a homodimer, chains A and B are discriminated by color (green and cyan, respectively). Each risk variant is identified by letters as well as colors, and TGMP is shown in stick-ball presentation. The 3-dimensional structure was drawn by using PyMOL software (accession code 5LPG of the Protein Data Bank, http://www.rcsb.org/pdb). (C) NUDT15 nucleotide diphosphatase activity was determined by using the PiPer Pyrophosphate Assay (Life Technologies) for each of the 3 novel variants, with wild-type (WT) and p.R139C proteins included as controls. (D) NUDT15 protein thermostability was measured by using SYPRO Orange (Molecular Probes) for each of the 3 variants, with wild-type and the p.R139C variant included as controls. The inflection point of each curve indicates the temperature for protein unfolding and is thus a measurement of stability. RFU, relative fluorescence unit.